Project Summary - Single-chain MspA for nanopore sequencing of DNA New DNA sequencing technologies have transformed biomedical research and clinical applications. Due to its low cost, fast sample processing time, minimal sample amount, and lack of sample amplification, nanopore sequencing is a very promising technology to further reduce costs and increase read length of the DNA sequencing. Essential to this method is a nanopore which can be produced from inorganic, organic or biological materials. Despite recent advancements significant challenges remain for nanopore sequencing of DNA. For example, the residual current of each nucleobase in the MspA pore is determined by approximately four nucleotides reducing the accuracy of base calling, DNA translocation through the pore is too fast, and the stochastic activity of DNA-processing enzymes complicates data analysis. These challenges could, in principle, be addressed by protein engineering, which, however, is hampered by the oligomeric structure of MspA and all other currently used biological nanopores. This proposal is based on the construction of single-chain MspA in which all eight subunits are linked. Single-chain MspA enables us to control the subunit assembly of the MspA pore and the chemical properties of each channel residue by site-directed mutagenesis. This technological breakthrough provides the basis for further improvement of the sequencing capabilities of MspA. The goals of this proposal are to characterize the channel activity of single-chain MspA and its DNA sequencing properties and to systematically alter the pore diameter of MspA by constructing scMspA pores with different subunit stoichiometries. This will enable us, for the first time, to examine the influence of the pore diameter on nucleobase recognition by a biological nanopore.